1. Field of the Invention
The present invention is directed to a method of stimulating superoxide generation using phenolic thioethers which stimulate the generation of superoxide.
2. Background Information and Related Art
Recently, oxygen radicals have been implicated in the pathogenesis of many diseases. This implication is reflected by the many conferences devoted to this topic, books on the subject of free radicals and disease, and the appearance of two new specialized journals: Free Radical Research Communications, and Free Radical Biology and Medicine.
Much is known about the physicochemical properties of the various oxygen radicals, but knowledge of their overall importance in the initiation and amplification of human disease is limited. Some clinical conditions in which oxygen radicals are thought to be involved are discussed in Cross, C. E., et al., "Oxygen Radicals and Human Disease," ANN. INT. MED., 107:526-545 (1987) (see Table 1, p. 527) and Ward, P. A., et al., "Oxygen Radicals, Inflammation, and Tissue Injury," FREE RADICAL BIOLOGY & MEDICINE, 5:403-408 (1988). Among the clinical conditions in which oxygen radicals are thought to be involved are, for example, inflammatory-immune injury, autoimmune diseases, ischemia-reflow states, aging disorders, cancer, cigarette-smoke effects, emphysema, acute respiratory distress syndrome (ARDS), atherosclerosis, rheumatoid arthritis, senile dementia, cataractogenesis, retinopathy of prematurity, and contact dermatitis.
Oxygen radicals are capable of reversibly or irreversibly damaging compounds of all biochemical classes, including nucleic acids, protein and free amino acids, lipids and lipoproteins, carbohydrates, and connective tissue macromolecules. These species may have an impact on such cell activities as membrane function, metabolism, and gene expression. Oxygen radicals are formed in tissues by many processes (see Cross, et al., p. 528, Table 2). These are believed to be both endogenous, such as mitochondrial, microsomal and chloroplast electron transport chains; oxidant enzymes such as xanthine oxidase, indoleamine dioxygenase, tryptophan dioxygenase, galactose oxidase, cyclooxygenase, lipoxygenase, and monoamine oxidase; phagocytic cells such as neutrophils, monocytes and macrophages, eosinophils, and endothelial cells; and antioxidation reactions; and exogenous, such as redoxcycling substances, drug oxidations, cigarette smoke, ionizing radiation, sunlight, heat shock and substances that oxidize glutathione. They may be involved in the action of toxins such as paraquat, cigarette smoke, and quinone antitumor drugs.
Generation of reactive oxygen species is a critical event in successful host defense against invading organisms. Both neutrophils and macrophages rely on a variety of oxidants to damage bacterial constituents (see V. L. Shepherd, "The role of the respiratory burst of phagocytes in host defense," SEMIN. RESPIR. INFECT. (United States) June 1986, 1(2) p. 99-106.
Various thioether compounds have been described previously. For example, U.S. Pat. No. 4,663,333 discloses compounds of the formula ##STR1## wherein: R.sub.1 and R.sub.2 are the same or different members of the group consisting of halo, phenyl, substituted phenyl and a ##STR2## group wherein n, m and p are independently an integer of from 1 to 8 provided n+m+p is equal to or less than 10; X is thio, sulfinyl or sulfonyl; Alk.sub.1 is straight or branched chain lower alkylene of 1 to 6 carbon atoms, R.sub.3 is lower alkyl, Alk.sub.2 is straight or branched chain alkylene of 1 to 4 carbon atoms; R.sub.4 is selected from the group consisting of hydrogen halo, hydroxy, lower alkyl and lower alkoxy; and the pharmaceutically acceptable salts there of. The compounds inhibit 5-lipoxygenase and are useful in the treatment of inflammation, allergy and hypersensitivity reactions and other disorders of the immune system.
Wagner, et al. U.S. Pat. No. 4,029,812, and related U.S. Pat. Nos. 4,076,841 and 4,078,084 which issued from divisional applications of the -812 application, all assigned to The Dow Chemical Company, disclose 2-(3,5-di-tert-butyl-4-hydroxyphenyl)thiocarboxylic acids, esters and simple amides which are hypolipidemics and are useful in reducing plasma lipid levels, especially cholesterol and triglyceride levels.
European Patent Application EP 0190685 discloses heterocyclic amides represented by the formula ##STR3## wherein R.sub.1 and R.sub.2 are the same or different members of the group consisting of halo, phenyl, substituted phenyl and a ##STR4## group wherein q, r and t are independently an integer of from 1 to 8 provided that q+r+t is equal to or less than 10; Y is thio, sulfinyl or sulfonyl; Alk is straight or branched chain lower alkylene, and R.sub.3 is a heterocyclic amine represented by the formula: ##STR5## wherein R.sub.4 is selected from the group consisting of hydrogen, lower alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, carboxyl or carboxyloweralkyl; X is selected from the group consisting of N--R.sub.4, O and CH.sub.2, m is 2 or 3; n is 2 or 3 when X is O or N--R.sub.4 and n is 1 to 3 when X is CH.sub.2 ; p is 0 to 2; and the pharmaceutically acceptable salts thereof. The compounds inhibit 5-lipoxygenase and are useful as anti-inflammatory and anti-allergy agents.
European Patent Application EP 0190682 discloses anilides represented by the formula ##STR6## wherein: R.sub.1 and R.sub.2 are the same or different members of the group consisting of halo, phenyl, substituted phenyl and a ##STR7## group wherein n, m and p are independently an integer of from 1 to 8 provided that n+m+p is equal to or less than 10; X is thio, sulfinyl or sulfonyl; Alk is straight or branched chain lower alkylene; R.sub.3 is hydrogen or lower alkyl; and R.sub.4 is phenyl or substituted phenyl. The compounds inhibit 5-lipoxygenase and are useful in the treatment of allergy and hypersensitiviy reactions and inflammation.
U.S. Pat. No. 4,857,588 discloses methods for inhibiting lipoxygenase and includes pharmaceutical formulations comprising a pharmaceutical carrier and an effective lipoxygenase inhibiting amount of a compound of the formula ##STR8## wherein: R.sub.1 and R.sub.2 are the same or different members of the group consisting of 1,1-dimethylethyl, halo, phenyl and substituted phenyl; Alk is straight or branched chain lower alkylene; R.sub.4 is hydrogen or lower alkyl; R.sub.3 is hydrogen or lower alkyl or a cycloalkyl group of from 3 to 8 carbon atoms. The disclosed compounds inhibit 5-lipoxygenase and are useful in the treatment of allergy and hypersensitivity reactions and inflammation.
United Kingdom Patent No. 1,557,622 discloses 3,5-di-tertiary-butyl-4-hydroxyphenyl pyridine compounds of the formula: ##STR9## or a pharmaceutically acceptable acid addition salt thereof, wherein: y is --O--, --S-- or --N(R.sup.4)-- [wherein R.sup.4 is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms (e.g. methyl, ethyl, propyl or butyl), an aralkyl group (e.g. benzyl, methoxybenzyl or phenethyl)];
each of R.sup.1 and R.sup.2 is a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 4 carbon atoms (e.g. methyl, ethyl, propyl or butyl); PA1 R.sup.3 is a hydrogen atom, a hydroxymethyl group or a group of the formula: ##STR10## m is 0 or 1; and n is 0, 1, 2 or 3. PA1 R.sub.1 stands for a hydrogen atom or a lower alkyl radical, PA1 R.sub.2 stands for one of the groups, ##STR12## CN, and, in case n equal 0 and R.sub.1 is a hydrogen, it can only be C.sub.6 H.sub.5 and in case n equal 1, it can also be OH, whereby R.sub.3 stands for an alkyl radical with 1 to 5 carbon atoms and R.sub.4 and R.sub.5, which may be the same or different, stand for hydrogen atoms, lower or medium alkyl radicals or, together with the nitrogen atom, stand for a ring that may contain another heteroatom, PA1 n stands for 0 or 1 as well as with the corresponding phenolates. The phenols are said to have biocidal activity but they are said to be above all suitable as intermediates for the preparation of biocidal substances, for example, phosphate esters and carbamates. PA1 Alk.sup.1 represents straight or branched chain alkylene of 1 to 10 carbon atoms; and PA1 R represents: PA1 a) ##STR14## wherein R.sup.3 is hydrogen or alkyl of 1 to 4 carbon atoms, Alk.sup.2 is straight or branched chain alkylene of 1 to 4 carbon atoms, and R.sup.4 is hydrogen or alkyl of 1 to 4 carbon atoms; PA1 b) ##STR15## wherein R.sup.3 is hydrogen or alkyl of 1 to 4 carbon atoms and R.sup.5 is phenyl or substituted phenyl; PA1 c) ##STR16## wherein R.sup.6 is a heterocyclic amine represented by the formula ##STR17## wherein R.sup.7 is hydrogen or lower alkyl and R.sup.8 is hydrogen, lower alkyl, phenyl, substituted phenyl, benzyl or substituted benzyl; or PA1 d) ##STR18## wherein R.sup.9 is hydrogen or alkyl of 1 to 4 carbon atoms and R.sup.10 is alkyl of 1 to 10 carbon atoms; PA1 Inhibition of 5-lipoxygenase, in vitro: PA1 The 100,000.times.g supernatant fraction of Rat Basophilic Leukemia Cell Homogenate (RBL-1) serves as a 5-lipoxygenase enzyme source. The enzyme is incubated with [1-.sup.14 C)-arachidonic acid and Ca++ in the presence and absence of test compound. The product of 5-lipoxygenase, 5-hydroxyeicosatetraenoic acid (5-HETE), is separated by thin-layer chromatography and measured by radioactivity. A compound inhibiting 5-HETE synthesis by 30% or more is considered active at that concentration. Initial screening doses are 1.times.10.sup.-4 M. When the compound inhibits more than 50% of 5-HETE synthesis at 10.sup.-4 M, that compound is tested at multiple dose levels to determine the IC.sub.50 value (inhibitory concentration to inhibit 50%). PA1 Human neutrophil superoxide generation: Superoxide generation by formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils was quantitated by the reduction of cytochrome C (Badwey, J. A., Curnutte, J. T. and Karnovsky, M. L., cis-Polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils. J. Biol. Chem. 256: 12640-12643, 1981.) To 5 million neutrophils in 2.85 ml of KrebsRinger phosphate buffer, pH 7.2, 50 ul of inhibitor (in 10% DMSO/buffer), and 50 ul ferricytochrome C (5 mM, stock) were added and preincubated for 3 minutes at 37.degree. C. Absorption measurements at 550 nm were recorded at start of preincubation. Fifty ul FMLP (6 uM, stock) was added to initiate reaction. A plateau was reached within 3 minutes and this reading--initial reading (before addition of FMLP) was used to calculate nanomoles of superoxide generated based on a molar extinction coefficient of 2.1.times.10.sup.4 cm.sup.-1 mole.sup.-1. PA1 Isolation of human neutrophils: Human neutrophils were isolated from freshly drawn blood of healthy donors. Two ml of 5% dextran (MW 200,000-300,000) in saline was added to 10 ml aliquots of blood, mixed and placed upright for 45 min. at 37.degree. C. Approx. 8-10 ml of the plasma-white cell suspension from the dextran sedimentation was layered on 3 ml of Ficol-paque in a 15 ml tube and centrifuged at 400 g for 30 min. The supernate, containing plasma and platelets, was discarded by aspiration, and the pellet, containing predominantly neutrophils, was resuspended in 1 ml saline. The suspension was transferred to a clean tube, and pooled with other aliquots of blood treated similarly. The pooled suspension was centrifuged at 350 g for 5 min. and supernate discarded. The pellet was resuspended in 5 ml of 0.05% NaCl with a plastic Pasteur pipette for 25 seconds to lyse contaminating red cells, then 5 ml of 1.75% NaCl added to regain isotonicity. The red cell lysing procedure was repeated, the cells suspended in appropriate buffer (depending on assay) and counted.
Preferable compounds of the formula are those wherein Y is --O-- or --N(R.sup.4)-- (wherein R.sup.4 is as defined above). These compounds are said to have antiatherosclerotic, antihyperlipidemic, cerebral vasodilating and antithrombotic activities, and are useful as drugs for the treatment of eschemic vascular diseases in mammals such as atherosclerosis, cardiac infarction, angina pectoris, cerebral infarction, cerebral hemorrhage, renal infarction, intermittent claudication, transient cerebral attack or thrombosis.
German Offenlegunsschrift 1 936 463 discloses phenols having the formula ##STR11## in which X and Y, which may be the same or different, stand for hydrogen or halogen atoms or lower alkyl radicals,